These experiments have been initiated to characterize embryonic stem cells, especially human embryonic stem cells (hESC), for studies of human dopaminergic neuronal function. In spite of the great importance of dopaminergic neurons for drug abuse, in addition to their therapeutic potential via transplantation, there have been enormous obstacles to the direct study of human dopaminergic neurons. Until very recently such neurons have been obtainable only from human fetal material. The advent of human ES cells has made the derivation human dopaminergic neurons in vitro in unlimited quantities for research use a possibility. Our initial experiments were focused on mouse ES cells, and promoter systems which could be employed for transgene expression. It was found that the CMV and especially the EF1 promoter led to long-term stable transgene expression in ES cells. Interestingly, expression via both the EF1 and CMV promoter disappeared as the ES cells differentiated into dopaminergic neurons, using an established system for dopaminergic differentiation by stromal cell-mediated differentiation. In addition, promoters derived from the tyrosine hydroxylase and GAD65 genes are now being investigated. Since the aim of these studies involved primarily the use of human ES cells, and there was very little data in the literature concerning characterization of most of the lines in the NIH stem cell registry, we therefore determined to characterize the general properties of the hESC lines from BresaGen Inc., in terms of pluripotency, differentiation, stable maintenance, and gene expression patterns. Small and large scale oligonucleotide and cDNA arrays were also employed to characterize gene expression patterns in undifferentiated hESC. Experiments were also undertaken to obtain dopaminergic neuronal differentiation from hESC. It was found that co-culture with the stromal PA6 cell line induced dopaminergic differentiation with a defined and reproducible time course. Cells positive for tyrosine hydroxylase were first detected after 10 days of co-culture, with maximal numbers of cells positive for tyrosine hydroxylase being present after 20-23 days of co-culture. Differentiated dopaminergic neurons expressed a number of markers for mature dopaminergic neurons, including transcripts characteristic of neurotransmitter function and response to growth factors. Most colonies in each culture were found to contain dopaminergic cells after differentiation, although a minority of cells within each colony were dopaminergic and other cell types including non-CNS cells (e.g., cells positive for smooth muscle actin) were also present. After transplantation into the brain, some dopaminergic neurons were found to survive, although numbers of non-neuronal cells (e.g., smooth muscle actin positive cells) were much larger than numbers of CNS cells. Therefore, significant improvements will be needed before dopaminergic neurons derived from hESC can be employed for therapeutic transplantation. In addition, a number of experiments were performed to identify the factors which are responsible for stromal cell-mediated differentiation. Factors identified as possible contributors include hepatocyte growth factor and FGF8; however, these factors alone are not sufficient to induce dopaminergic differentiation. We have also identified a variant hESC line, BG01V, with karyotypic abnormalities, which can be grown more easily than the normal BG01 hESC line, but which undergoes dopaminergic differentiation with the same pattern and time course as BG01. This cell line may be very useful for identifying the factors responsible for stromal cell-mediated dopaminergic differentiation. Dopaminergic neurons derived from hESC were found to be sensitive to the dopaminergic neurotoxin MPP+, providing a cellular model through which neuroprotective agents can be tested in human cells. The neurotrophic factor GDNF was found to be protective against MPP+ toxicity, and increased expression of the dopaminergic transcription factor Nurr 1. We have recently also completed a comprehensive characterization of gene expression in neural stem cells and dopaminergic neurons during differentiation from hESC using the Massive Parallel Signature Sequencing (MPSS) technique. For these experiments, differentiating dopaminergic neurons were isolated by fluorescent-activated cell sorting on the basis of polysialyated N-CAM expression on cell surfaces. These sorted cells were found to be capable of complete differentiation, as indicated by neuronal function defined by electrophysiological criteria. Current experiments are focused on developing genetically altered cell lines for specific purposes, such as monitoring the status of differentiation via reporter transgenes, and employing hESC-derived dopaminergic neurons to characterize the effects of drugs of abuse on human dopaminergic neuronal function. In addition, alternative protocols for producing differentiated human neurons from hESC are being developed; including testing effects of differentiation-promotion agents and alternative protocols for cell-mediated differentiation. These alternative differentiation protocols can be used to produce cell types which are useful for studies of drugs of abuse on specific human neuronal phenotypes and also for neuronal transplantation protocols.